This application claims benefit of Japanese Application No. Hei 11-258373 filed in Japan on Sep. 13, 1999, the contents of which are incorporated by this reference.
These inventions relate to methods and apparatus for observation using quantum dot. More particularly, they relate to emission observation methods using quantum dots and also relate to emission observation apparatus ofr carrying out the emissio observation methods.
Ultrahigh-sensitivity detecting machinery and materials recently developed have made it possible to detect and identify single molecules and to observe the motion thereof and have played an important role in analytical chemistry, molecular biology and the analysis of nanostructures.
In most conventional methods of observing single molecules, target molecules are labeled with fluorescent dyes and observed with a fluorescence miocroscope. However, as a substitute for the conventional methods, a method in which a sample is labeled with quantum dots has recently been proposed by W. C. W. Chan et al. (W. C. W. Chan and S. Nie, Science, vol. 281 (1998), p. 2016).
A typical example of quantum dot structures shown by W. C. W. Chan et al. is illustrated in FIG. 3. That is, the surface of a semiconductor CdSe microsphere with a diameter of 2 to 5 nanometers is coated with ZnS, and hydroxyl groups are adsorbed to the ZnS coating through sulfur molecules. One of the hydroxyl groups is coupled to a target protein.
In comparison to those of the conventional fluorescent dyes, as shown in FIG. 4, the emission characteristics of quantum dots have the following features. The half-width of the emission spectrum is about {fraction (1/20)} of the center wavelength, which is as narrow as about ⅓ of that of fluorescent dyes. The peak wavelength of the emission spectrum can be set relatively freely within a range of from about 400 nanometers to about 2000 nanometers by selecting a size and material of quantum dots. Further, the excitation spectrum increases in intensity as the wavelength becomes shorter in the visible to ultraviolet regions irrespective of the position of the center wavelength of the emission spectrum. FIG. 4 shows excitation and emission spectral distributions of quantum dots of CdSe and InP that are different in particle diameter from each other.
When used for single-molecule detection, quantum dots have the following advantages in comparison to conventional fluorescent dyes of organic substances. One of the advantages is that the size is very small so that the quantum dots can hardly interfere with the motion of the target molecule. Another advantage is that the emission efficiency is much higher than that of the conventional fluorescent dyes, and it is therefore possible to detect single molecules with high sensitivity. Another advantage is that the toxicity is very low; therefore, in vivo observation can be performed. Still another advantage is that fading can hardly occur. In view of these advantages, quantum dots are expected to be widely used in future analyses using the technique of single-molecule detection in place of the conventional fluorescent dyes.
In simultaneous identification of a plurality of different kinds of molecules, quantum dots have the following advantages in comparison to the conventional fluorescent dyes. With quantum dots, a plurality of emission center wavelengths can be set relatively freely by selecting a particle size and a material as has been stated above. Moreover, the half-width of the emission spectrum is narrow. Therefore, it is possible to identify a larger number of different kinds of molecules than in the case of the conventional method using fluorescent dyes in a usable wavelength band. Further, in the case of quantum dots, the excitation spectrum increases in excitation intensity as the wavelength becomes shorter in the visible region irrespective of the position of the center wavelength of the emission spectrum. Therefore, it is possible to efficiently excite all quantum dots in a single wavelength band.
Incidentally, an ordinary incident light fluorescence microscope can be used to identify a plurality of different kinds of substances labeled with quantum dots.
An ordinary incident light fluorescence microscope has the following optical elements: an excitation filter for selecting a wavelength band for excitation of a fluorescent dye used; a barrier filter that blocks light in the transmission wavelength band of the excitation filter and passes light in the fluorescence wavelength band; and a dichroic mirror that reflects light in the transmission wavelength band of the excitation filter and passes light in the fluorescence wavelength band. Light from a light source passes through the excitation filter to become excitation light limited to a predetermined wavelength band. The excitation light is reflected by the dichroic mirror toward a sample in a coaxial relation to an observation light path to illuminate the sample. Fluorescence from the sample excited by the excitation light travels along the observation light path in the opposite direction to the excitation light and passes through the dichroic mirror. The barrier filter blocks the residual component of the excitation light in the light passing through the dichroic mirror and passes the fluorescent light. By using these three optical elements, it is possible to produce fluorescence by excitation light and to detect fluorescent light, which is feeble in comparison to the excitation light, with high contrast.
In the ordinary incident light fluorescence microscope, the above-described three optical elements are integrated into a fluorescence cube, and a multiplicity of fluorescence cubes adapted for the emission characteristics of various fluorescent dyes are prepared. The fluorescence cubes are often arranged to be switched from one to another by a simple operation according to each particular fluorescent dye used. For example, there is a publicly known incident light fluorescence microscope arrangement in which a plurality of fluorescence cubes are placed on a turret so that the fluorescence cubes can be switched from one to another simply by rotating the turret. There is also a known arrangement in which a plurality of fluorescence cubes are placed on a slider so as to be switchable from one to another simply by sliding the slider.
To observe a sample labeled with a plurality of quantum dots different in size or material from each other by using such an incident light fluorescence microscope, the wavelength characteristics of the three optical elements should be set as shown in FIG. 5. That is, the excitation filter blocks light of longer wavelength than the minimum value xcex0 in the emission band of quantum dots used and passes light of shorter wavelength than the minimum value xcex0. The barrier filter blocks light of shorter wavelength than xcex0 and passes light of longer wavelength than xcex0. The dichroic mirror reflects light of shorter wavelength than xcex0 and passes light of longer wavelength than xcex0. By setting the wavelength characteristics of the three optical elements as stated above, feeble light emitted from quantum dots can be observed with high contrast without being superimposed on the intense excitation light.
It is preferable from the viewpoint of detection efficiency to prepare a plurality of fluorescence cubes different in xcex0 and to arrange them to be switchable from one to another freely according to the emission band of quantum dots used.
Incidentally, when an ordinary incident light fluorescence microscope is used to detect a sample labeled with quantum dots, the following problems arise:
If it is intended to excite quantum dots efficiently and increase the intensity of light emitted from the quantum dots so as to detect the light with high contrast, it is preferable that the wavelength region of excitation light that is shorter than xcex0 should be widened as much as possible. However, the short-wavelength component of excitation light may cause autofluorescence to occur from an encapsulating medium fixing the sample and also from a vitreous material constituting the objective lens. The autofluorescence may cause the contrast of the observation image to be reduced remarkably. Because the degree of autofluorescence varies according to the encapsulating medium and the objective lens material, it is not preferable to preset an excitation wavelength band. That is, if an excitation wavelength band is set at a certain value in advance, the emission efficiency of quantum dots will not be optimized when a sample and an objective lens that can hardly emit autofluorescence are used. In particular, when extremely feeble light is to be detected as in single-molecule observation, the detection capability will be reduced remarkably. In addition, xcex0 for the detection of such extremely feeble light may vary according to the sample and the objective lens used.
In view of the above-described problems with the prior art, the inventions described/claimed herein provide methods and systems suitable for the observation of quantum dots.
The inventions provide observation methods wherein excitation light is applied to quantum dots, and light emitted from the quantum dots in a plurality of different wavelength bands is detected. The wavelength band of the excitation light is variably adjustable.
According to these inventions, the wavelength band of excitation light is adjusted according to the characteristics of autofluorescence that may occur in an optical system used, thereby allowing the quantum dot excitation efficiency to be maximized within a range in which the fluorescence observation is not affected by the autofluorescence. Therefore, the observation method is particularly suitable for the detection of feeble light as in single-molecule observation.
Other features and advantages of the inventions will be apparent from the specification.
The inventions accordingly comprise the features of construction, combinations of elements, and arrangement of parts which will be exemplified in the construction hereinafter set forth, and the scope of the inventions will be indicated in the claims.